Ana Diaz

Inhibition of xanthine oxidase by allopurinol prevents skeletal muscle atrophy: role of p38 MAPKinase and E3 ubiquitin ligases.

Alterations in muscle play an important role in common diseases and conditions. Reactive oxygen species (ROS) are generated during hindlimb unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of this study was to determine the mechanism by which XO activation causes unloading-induced muscle atrophy in rats, and its possible prevention by allopurinol, a well-known inhibitor of this enzyme. For this purpose we studied one of the main redox sensitive signalling cascades involved in skeletal muscle atrophy i.e. p38 MAPKinase, and the expression of two well known muscle specific E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFbx; also known as atrogin-1) and Muscle RING (Really Interesting New Gene) Finger-1 (MuRF-1). We found that hindlimb unloading induced a significant increase in XO activity and in the protein expression of the antioxidant enzymes CuZnSOD and Catalase in skeletal muscle. The most relevant new fact reported in this paper is that inhibition of XO with allopurinol, a drug widely used in clinical practice, prevents soleus muscle atrophy by ∼20% after hindlimb unloading. This was associated with the inhibition of the p38 MAPK-MAFbx pathway. Our data suggest that XO was involved in the loss of muscle mass via the activation of the p38MAPK-MAFbx pathway in unloaded muscle atrophy. Thus, allopurinol may have clinical benefits to combat skeletal muscle atrophy in bedridden, astronauts, sarcopenic, and cachexic patients.

Life-long spontaneous exercise does not prolong lifespan but improves health span in mice

BackgroundLife expectancy at birth in the first world has increased from 35 years at the beginning of the 20th century to more than 80 years now. The increase in life expectancy has resulted in an increase in age-related diseases and larger numbers of frail and dependent people. The aim of our study was to determine whether life-long spontaneous aerobic exercise affects lifespan and healthspan in mice.ResultsMale C57Bl/6J mice, individually caged, were randomly assigned to one of two groups: sedentary (n = 72) or spontaneous wheel-runners (n = 72). We evaluated longevity and several health parameters including grip strength, motor coordination, exercise capacity (VO2max) and skeletal muscle mitochondrial biogenesis. We also measured the cortical levels of the brain-derived neurotrophic factor (BDNF), a neurotrophin associated with brain plasticity. In addition, we measured systemic oxidative stress (malondialdehyde and protein carbonyl plasma levels) and the expression and activity of two genes involved in antioxidant defense in the liver (that is, glutathione peroxidase (GPx) and manganese superoxide dismutase (Mn-SOD)). Genes that encode antioxidant enzymes are considered longevity genes because their over-expression may modulate lifespan. Aging was associated with an increase in oxidative stress biomarkers and in the activity of the antioxidant enzymes, GPx and Mn-SOD, in the liver in mice. Life-long spontaneous exercise did not prolong longevity but prevented several signs of frailty (that is, decrease in strength, endurance and motor coordination). This improvement was accompanied by a significant increase in the mitochondrial biogenesis in skeletal muscle and in the cortical BDNF levels.ConclusionLife-long spontaneous exercise does not prolong lifespan but improves healthspan in mice. Exercise is an intervention that delays age-associated frailty, enhances function and can be translated into the clinic.

Right ventricular involvement in anterior myocardial infarction: a translational approach

AIMS The aim of the present study was to evaluate the involvement of the right ventricle (RV) in reperfused anterior ST-elevation myocardial infarction (STEMI). METHODS AND RESULTS Left anterior descending (LAD)-perfused area (using thioflavin-S staining after selective infusion in proximal LAD artery, %), infarct size (using triphenyltetrazolium chloride staining, %), and salvaged myocardium (% of LAD-perfused area) in the right and left ventricle (LV) were quantified in a 90-min LAD occlusion 3-day reperfusion model in swine (n = 8). Additionally, we studied, using cardiovascular magnetic resonance, 20 patients with a first STEMI due to proximal LAD occlusion treated with primary angioplasty. Area at risk (T2-weighted sequence, %), infarct size (late enhancement imaging, %), and salvaged myocardium (% of area at risk) in the right and LV were quantified. In swine, a large LAD-perfused area was detected both in the right and LV (30 +/- 5 vs. 62 +/- 15%, P< 0.001) but more salvaged myocardium (94 +/- 6 vs. 73 +/- 11%, P< 0.001) resulted in a smaller right ventricular infarct size (2 +/- 1 vs. 16 +/- 5%, P< 0.001). Similarly, in patients a large area at risk was detected both in the right and LV (34 +/- 13 vs. 43 +/- 12%, P = 0.02). More salvaged myocardium (94 +/- 10 vs. 33 +/- 26%, P < 0.001) resulted in a smaller infarct size (2 +/- 3 vs. 30 +/- 16%, P< 0.001) in the RV. CONCLUSION In reperfused extensive anterior STEMI, a large area of the RV is at risk but the resultant infarct size is small.

Human CD133+ bone marrow-derived stem cells promote endometrial proliferation in a murine model of Asherman syndrome

OBJECTIVE To investigate the engraftment and proliferation of superparamagnetic iron oxide nanoparticles (SPIOs)-labeled human CD133(+) bone marrow-derived stem cells (BMDSCs) in an animal model of Asherman syndrome (AS). DESIGN Prospective experimental animal study. SETTING University research laboratories. ANIMAL(S) Nonobese diabetic mice (strain code 394; NOD.CB17- Prkdc(scid)/NcrCrl) in which AS was induced according to a published protocol. INTERVENTION(S) Human CD133(+) BMDSCs were obtained from patients undergoing autologous cell therapy in refractory AS and endometrial atrophy, labeled with SPIOs and injected either intrauterinely (n = 5) or systemically through the tail vein (n = 5) in the animal model. MAIN OUTCOME MEASURE(S) Accumulation of collagen and glycosaminoglycan deposits detected by trichrome staining. Percentage and localization of engrafted human SPIOs-labeled CD133(+) BMDSCs by Prussian blue staining. Cell proliferation assay using Ki67 and reverse transcriptase-polymerase chain reaction (PCR) for specific paracrine factors. RESULT(S) The induction of the AS in the murine model was demonstrated by the accumulation of collagen and glycosaminoglycan deposits in the damaged horns by trichrome staining. Human SPIOs labeled CD133(+) BMDSCs homing represents 0.59% and 0.65% of total number of cells present in the horns after intrauterine or tail vein injections, respectively. Engrafted cells were localized around endometrial blood vessels, inducing proliferation in surrounding cells based on Ki67 and regulation of the paracrine factors thrombospondin 1 and insulin-like growth factor 1. CONCLUSION(S) The injection of human SPIOs labeled CD133(+) BMDSCs in a murine model of AS confirms that these cells engraft around endometrial vessels, inducing proliferation of surrounding cells through paracrine molecules such as thrombospondin 1 and insulin-like growth factor 1. CLINICAL TRIAL REGISTRATION NUMBER NCT02144987.

Effect of ischemic postconditioning on microvascular obstruction in reperfused myocardial infarction. Results of a randomized study in patients and of an experimental model in swine.

BACKGROUND Ischemic postconditioning (PCON) appears as a potentially beneficial tool in ST-segment elevation myocardial infarction (STEMI). We evaluated the effect of PCON on microvascular obstruction (MVO) in STEMI patients and in an experimental swine model. METHODS A prospective randomized study in patients and an experimental study in swine were carried out in two university hospitals in Spain. 101 consecutive STEMI patients were randomized to undergo primary angioplasty followed by PCON or primary angioplasty alone (non-PCON). Using late gadolinium enhancement cardiovascular magnetic resonance, infarct size and MVO were quantified (% of left ventricular mass). In swine, using an angioplasty balloon-induced anterior STEMI model, MVO was defined as the % of area at risk without thioflavin-S staining. RESULTS In patients, PCON (n=49) in comparison with non-PCON (n=52) did not significantly reduce MVO (0 [0-1.02]% vs. 0 [0-2.1]% p=0.2) or IS (18 ± 13% vs. 21 ± 14%, p=0.2). MVO (>1 segment in the 17-segment model) occurred in 12/49 (25%) PCON and in 18/52 (35%) non-PCON patients, p=0.3. No significant differences were observed between PCON and non-PCON patients in left ventricular volumes, ejection fraction or the extent of hemorrhage. In the swine model, MVO occurred in 4/6 (67%) PCON and in 4/6 (67%) non-PCON pigs, p=0.9. The extent of MVO (10 ± 7% vs. 10 ± 8%, p=0.9) and infarct size (23 ± 14% vs. 24 ± 10%, p=0.8) was not reduced in PCON compared with non-PCON pigs. CONCLUSIONS Ischemic postconditioning does not significantly reduce microvascular obstruction in ST-segment elevation myocardial infarction. Clinical Trial Registration http://www.clinicaltrials.gov. Unique identifier: NCT01898546.

Naked DNA delivery to whole pig cardiac tissue by coronary sinus retrograde injection employing non-invasive catheterization

Hydrodynamic injection has demonstrated to be very efficient in the liver of small animals, although this procedure must be translated to the clinical practice in a milder but no less efficient way. The present study evaluates the capacity of non‐invasive interventional catheterization as a procedure for naked DNA delivery to the heart in large animals.

Overexpression of the truncated form of High Mobility Group A proteins (HMGA2) in human myometrial cells induces leiomyoma-like tissue formation

The pathogenesis of uterine leiomyomas, the most common benign tumor in women, is still unknown. This lack of basic knowledge limits the development of novel non-invasive therapies. Our group has previously demonstrated that leiomyoma side population (SP) cells are present in tumor lesions and act like putative tumor-initiating stem cells in human leiomyoma. Moreover, accumulated evidence demonstrates that these benign tumors of mesenchymal origin are characterized by rearrangements of the High Mobility Group A proteins (HMGA). In this work, we tested the hypothesis that leiomyoma development may be due to overexpression of HMGA2 (encoding high mobility group AT-hook2) in myometrial stem cells using in vitro and in vivo approaches. Our work demonstrates that the truncated/short form of HMGA2 induces myometrial cell transformation toward putative tumor-initiating leiomyoma cells and opens up new possibilities to understand the origin of leiomyomas and the development of new therapeutic approaches.

Microvascular obstruction in the right ventricle in reperfused anterior myocardial infarction. Macroscopic and pathologic evidence in a swine model

INTRODUCTION Data on right ventricular (RV) involvement in anterior myocardial infarction are scarce. The presence of RV microvascular obstruction (MVO) in this context has not been analyzed yet. The aim of the present study was to characterize the presence of MVO in the RV in a controlled experimental swine model of reperfused anterior myocardial infarction. MATERIALS AND METHODS Left anterior descending (LAD) artery-perfused area (thioflavin-S staining after selective infusion in LAD artery), infarct size (lack of triphenyltetrazolium-chloride staining) and MVO (lack of thioflavin-S staining in the core of the infarcted area) in the RV were studied. A quantitative (% of the ventricular volume) and semiquantitative (number of segments involved) analysis was carried out both in the RV and LV in a 90-min left anterior descending balloon occlusion and 3-day reperfusion model in swine (n=15). RESULTS RV infarction and RV MVO (>1 segment) were detected in 9 (60%) and 6 (40%) cases respectively. Mean LAD-perfused area, infarct size and MVO in the RV were 33.8 ± 13%, 13.53 ± 11.7% and 3.4 ± 4.5%. Haematoxylin and eosin stains and electron microscopy of the RV-MVO areas demonstrated generalized cardiomyocyte necrosis and inflammatory infiltration along with patched hemorrhagic areas. Ex-vivo nuclear magnetic resonance (T2 sequences) microimaging of RV-MVO showed, in comparison with remote non-infarcted territories, marked hypointense zones (corresponding to necrosis, inflammation and hemorrhage) in the core of hyperintense regions (corresponding to edema). CONCLUSIONS In reperfused anterior myocardial infarction, MVO is frequently present in the RV. It is associated with severe histologic repercussion on the RV wall. Nuclear magnetic resonance appears as a promising technique for the noninvasive detection of this phenomenon. Further studies are warranted to evaluate the pathophysiological and clinical implications.

A Multidisciplinary Assessment of Remote Myocardial Fibrosis After Reperfused Myocardial Infarction in Swine and Patients.

In extensive nonreperfused myocardial infarction (MI), remote fibrosis has been documented. Early reperfusion by primary angioplasty represents the gold standard method to minimize the extension of the infarction. We aimed to ascertain whether fibrosis also affects remote regions in reperfused MI in swine and patients. Swine were subjected to a transient occlusion of the left anterior descending artery followed by 1-week or 1-month reperfusion. Collagen content in the remote area macroscopically, microscopically, by magnetic resonance microimaging, and at the molecular level was similar to controls. In patients with previous MI, samples from autopsies displayed a significant increase in collagen content only in the infarct region. In patients with previous MI submitted to cardiac magnetic resonance-T1 mapping, the extracellular volume fraction in remote segments was similar to that for controls. In all scenarios, the remote region did not show a significant increase of collagen content in comparison with controls.

Human Interleukin-10 Naked DNA Delivery to Infarcted Pig Heart by Catheter Mediated Retrograde Injection in Coronary Sinus

Background: Interleukin-10 (IL-10) has proved to be important in recovery after acute myocardial infarction; increasing its expression in infarcted or bystander tissue therefore could be of great importance. Hydrodynamic DNA injection has been found to be very efficient in transferring genes to the liver of small animals, but the procedure is very aggressive and must be made compatible with clinical practice in a milder but not less efficient way. The present work evaluates the efficiency of noninvasive catheterization of the coronary sinus for human IL-10 gene transfer to infarcted and non-infarcted pig heart, with therapeutic production of the human protein. Methods: Myocardial infarction was induced in pigs by a catheter-based approach to occlude the left anterior descending artery. After myocardial infarction verification, two catheters were placed in the coronary sinus, one of them to block blood circulation and the other to retrogradely inject 50 ml of a saline solution of DNA (20 μg/ml) containing the hIL-10 gene, and testing different flow rate conditions (control, 2, 5 and 10 ml/s). Results: Therapeutic levels of hIL-10 protein were found in coronary sinus blood 2 and 72 hours after cathetermediated hydrofection at 5 and 10 ml/s flow rate. Molecular analyses to evaluate the delivered DNA, its transcription to RNA and translation were also performed, and data were expressed as copies per cell. Conclusion: Catheter-mediated gene transfer through the coronary sinus is a mild and well-tolerated procedure that achieves protective hIL-10 protein levels, which could minimize the inflammatory response after myocardial infarction.